1. Field of the Invention
This invention relates to a novel method for a two-site immunoassay.
2. Description of the Prior Art
Two-site immunoradiometric assays for antigens which can bind to at least two antibodies are known. Those known two-site immunoradiometric assays involve a first incubation of unlabeled antibody coupled to an insoluble matrix with the fluid containing the antigen having more than one antigenic determinant, followed by a second incubation with a labeled antibody. Woodhead, Addison and Hales, Br. Med. Bull., Vol. 39, No. 1, 44-49 (1974); Abraham (ed.), Handbook of Radioimmunoassay, Ch. 4 (Marcel Dekker, Inc., 1977).
Such two-site immunoradiometric assays are discussed in Radioimmunoassay And Related Procedures In Medicine, Vol. 1, 131-47, 149-64 (International Atomic Energy Agency, Vienna 1974).
Corning Medical Diagnostics has marketed an assay for determination of human thyroid stimulating hormone (hereinafter referred to as "TSH"). See Corning, TSH (.sup.125 I) Radioimmunoassay Test System (March, 1977). In that assay as described by Corning, the radioactive, .sup.125 I antibody, specific for TSH, is added to a known amount of TSH standard or to a patient serum sample. The radioactive, .sup.125 I antibody will bind to any TSH present and form a labeled complex. After a first incubation of equilibration period, another antibody, also raised against TSH, is added to the reaction mixture. This antibody has been covalently bonded to microscopic glass particles and serves to bind the labeled complex formed in the first step. After binding separation can occur by simple centrifugation. The precipitated complex is counted for .sup.125 I radioactivity. A standard curve is constructed in which radioactivity bound to glass is plotted against TSH concentration. The counts bound in the presence of unknown patient samples are compared to the standard curve and the appropriate TSH concentration is determined by interpolation.
Corning also markets an assay for the determination of human thyroxine-binding globulin ("TBG"). See Corning, TBG .sup.125 I Radioimmunoassay Test System (December, 1977). In that assay, an immobilized antibody specific for TBG is added to a known amount of TBG standard or to a patient serum sample. After vortexing, radioactive .sup.125 I labeled thyroxine (".sup.125 I T4") is added to the reaction mixture. According to Corning, the .sup.125 I T4 will then partition between the binding sites on TBG and bovine serum albumin molecules present in the buffer. The TBG can thus become bound both to the immobilized antibody and to the .sup.125 I T4.
The use of antibodies covalently bound to a solid-phase in radioimmunoassays for proteins and polypeptides is shown by U.S. Pat. No. 3,555,143, issued Jan. 12, 1971, to Axen and Wide. Pharmacia has applied that method in a commercial radioimmunoassay for TSH. In this procedure, the antibody is covalently bound to a polydextran. The sample to be determined and the solid-phase antibody are incubated at room temperature for 18-24 hours followed by addition of radioactively labeled TSH and further incubation with agitation for 18-24 hours. The solid phase is separated, washed three times and its radioactivity measured.
The patent application by Monthony et al. Ser. No. 621,197, filed Oct. 9, 1975, describes an immunofluorescent assay method in which the immune reactants are covalently bound to water insoluble hydrophilic polymeric particles of about 0.1-10 microns in size. The solid phase antibody is mixed with the sample containing antigen (or hapten) to be determined and corresponding antigen (or hapten) which has been fluorescently labeled, so as to bind a quantity of labeled and unlabeled antigen. The solid phase is separated and the fluorescence measured by optical spectroscopy, the concentration of the unknown immune reactant being a function of the value of the fluorescence. This application also describes a two-site immunoassay method in which an excess of solid phase antibody is first reacted with the sample containing antigen (or hapten) to be determined, and the complex is subsequently reacted with an excess of fluorescently labeled antibody.